Mitochondria produce virtually all of the energy supply in tissues with high energy demands, and many diseases of impaired mitochondrial function have been described involving both mitochondrial and nuclear genomes (B. Robinson, Journal of Bioenergetics & Biomembranes 26:311-316 (1994); D. Wallace, American Journal of Human Genetics 57:201-223 (1995)). Of the hundreds of proteins that are found within mitochondria, the mitochondrial genome encodes only 13 of these and the rest must be imported from the cytosol (R. Jansen, Human Reproduction 15 Suppl 2:1-10 (2000); M. Douglas,. and M. Takeda, Trends in Biochemical Sciences 10:192-194 (1985)). Based on in vitro observations, proteins targeted to the mitochondria are thought to be completely synthesized in the cytoplasm and cross the mitochondrial membranes post-translationally (G. Schatz, and B. Dobberstein, Science 271:1519-1526(1996)). Proteins that are nuclearly encoded and targeted to the inner and outer membranes, inter-membrane space, or matrix, are aided by the use of presequences at the N-terminus of the precursor protein (G. Schatz, Journal of Biological Chemistry 271:31763-31766 (1996); A. Mayer et al. Cell 80:127-137(1995)). Most of these mitochondrial targeting sequences (MTS) consist of 10 to 70 amino acids that are removed by 1 or 2 proteolytic steps once inside the mitochondria. At the outer mitochondrial membrane the MTS is recognized by a receptor complex (OM37, OM70, or an OM37/OM70 complex), the presequence is passed to OM20, or an OM20/OM22 complex, and membrane translocation proceeds through the channel in the outer membrane translocation machinery (TOM complex) (T. Komiya, and K. Mihara, Journal of Biological Chemistry 271:22105-22110 (1996)). After translocation of the presequence across the outer membrane, a portion of the presequence is recognized by a receptor of the inner membrane (IM) translocation machinery (TIM complex) (M. Bauer, et al., Cell 87:33-41 (1996)). The precursor protein then proceeds through both the TOM and TIM complexes in conjunction with mitochondrial HSP70 and Tim44 on the matrix side of the IM. Import is driven by ATP hydrolysis of the HSP70 motor, and the transit peptide is cleaved by the mitochondrial processing peptidase (G. Isaya, et al., Proceedings of the National Academy of Sciences of the United States of America 89:8317-8321 (1992); T. Omura, Journal of Biochemistry 123:1010-1016 (1998)) Finally, the protein is deposited in the matrix or integrated into the IM (J. Berthold, et al., Cell 81:1085-1093(1995); C. Ungermann, et al., EMBO Journal 15:735-744 (1996)).
Both nuclear-encoded and mitochondrial-encoded proteins can be mutated, deleted, or be insufficient in amount, leading to functional problems (D. Wallace, Scientific American 277:40-47 (1997); C. Graff, et al., Journal of Internal Medicine 246:11-23 (1999)). Furthermore, there is now a growing body of information on how the compartmentation of mitochondrial proteins, and their function, can be disturbed by acquired conditions, such as aging, oxidative stress, and ischemia, and which may lead to disease or decreased tissue function (J. Rosenblum, et al., Proceedings of the National Academy of Sciences of the United States of America 93:4471-4473(1996); G. Davey, et al., Journal of Biological Chemistry 273:12753-12757 (1998)). One possible way to alleviate these problems is to deliver exogenous protein to mitochondria to replace the defective or deficient proteins. To date this has been very difficult with viral or nonviral vectors (R. Owen and T. Flotte, Antioxidants & Redox Signaling 3:451-460 (2001); Y. Bai, et al., Journal of Biological Chemistry 276:38808-38813 (2001); K. Nakada, et al., Nature Medicine 7:934-940 (2001); V. Weissig, and V. Torchilin, Advanced Drug Delivery Reviews 49:127-149 (2001); B. Seo, et al., Proceedings of the National Academy of Sciences of the United States of America 95:9167-9171 (1998)). In particular, viral-mediated gene transfer has been associated with poor transfection rates and the risk of death in patients (P. Noguchi, N. Engl. J. Med. 348:193-194 (2003). Non-viral techniques, such as liposome mediated gene transfer, have been even more disappointing when applied to vertebrate tissues.
Mitochondria are organelles that are vulnerable to damage for at least three reasons. First; mitochondria contain a limited genome, approximately 16 kb in humans. Thus, the majority of proteins essential for continued function must be imported into mitochondria. Defects in these nuclear encoded proteins result in human disease. Furthermore, diseases involving mitochondrial-encoded DNA present a special challenge for potential gene therapy because most of the proteins encoded are hydrophobic and lack a transit peptide. Thus, these proteins are difficult to keep in the unfolded conformation needed for import. This means their ability to respond appropriately in synthesizing new proteins after damaging conditions such as birth asphyxia, heart attack, stroke, or aging, is limited. Second, mitochondria lack histones and thus, do not have efficient mechanisms for protection and repair of DNA damage. Consequently, mutations within the mitochondrial DNA (mtDNA) are cumulative and result in disease, such as Leber's Hereditary Optic Neuropathy. Third, mitochondria contain a highly oxidative environment and generate 95% of the total superoxide radicals in the cell (A. Boveris, Methods in Enzymology 105:429-435 (1984)). Thus, oxidative damage to the mtDNA and proteins is constant and certain. Mitochondria have evolved protective mechanisms against this damage, such as mitochondrial superoxide dismutase; however, these can be overwhelmed by abnormal physiology with resultant overproduction of superoxide and damaging free radicals.
Given that many human diseases involve mitochondrial dysfunction, and there are currently no satisfactory methods to correct these defects, there is a need to develop techniques to screen for these defects and therapies to correct mitochondrial defects.
Background on Protein Transduction Domains (PTD). Delivery of drugs and therapeutic compounds is primarily limited by their ability to penetrate the cell membrane. The bioavailability of compounds targeted to intracellular sites depends on the conflicting requirements of being sufficiently polar for administration and distribution, yet non-polar enough to diffuse through the non-polar lipid bilayer of the cell (D. Begley, Journal of Pharmacy & Pharmacology 48:136-146 (1996)). In addition, the molecular weight of most drugs that can easily traverse the lipid membrane is approximately 500 Da (V. Levin, Journal of Medicinal Chemistry 23:682-684 (1980)). Thus, most successful compounds have narrow physical characteristics. Many promising drugs fail because they fall outside of this range and efforts to make them available may be toxic. In addition to this, many sites of action for presumed therapeutic compounds, such as enzymes or regulatory proteins, require processing and targeting of the compound once inside the cell.
To address these problems, delivery of a gene product into cells has been heavily investigated using both viral and non-viral vectors, as well as naked DNA and liposome-mediated gene transfer (V. Geromel, et al., Antisense & Nucleic Acid Drug Development 11:175-180 (2001); S. Francis, et al., American Journal of PharmacoGenomics 1:55-66 (2001); B. Cao, et al., Microscopy Research & Technique 58:45-51 (2002)). However, drawbacks with current ‘gene therapies’, such as viral toxicity and inefficient transfection rates, the immune response to viral vectors, and difficulty in creating the gene vector, have limited their usefulness in gene therapy (M. Rebolledo, et al., Circulation Research 83:738-74 (1998); T. Ritter, et al., Biodrugs 16:3-10 (2002)). Furthermore, localizing a gene product within the cell has been difficult. For example, attempts to deliver proteins to mitochondria within the cells to correct defects in their function have been limited (P. Seibel, et al., Nucleic Acids Research 23:10-17 (1995)). The use of mitochondrial targeting sequences to localize fusion proteins within mitochondria is not a new concept. Fusion proteins made with mitochondrial signal sequences have been transfected into cultured cells and shown to not only be targeted to mitochondria, but also processed, allowing for complete localization and functionality of the fused protein (C. Zhang, et al., Biochemical & Biophysical Research Communications 242:390-395 (1998); B. Seaton, and L. Vickery, Archives of Biochemistry & Biophysics 294:603-608 (1992)). However, the transfer of this technology to tissues in vivo has not been successful, in part, because of problems with delivery of the gene product.
Recently, a novel strategy for delivery of synthetic compounds has been described and is being actively investigated by both industry and academic researchers (R. Service, Science 288:28-29 (2000)). Positively charged, cationic peptides, are known to cross cell membranes independent of receptors or specific transport mechanisms (S. Schwarze, et al., Science 285:1569-1572 (1999); A. Ho, et al., Cancer Research 61:474-477 (2001);. M. Morris, et al., Nature Biotechnology 19:1173-1176 (2001); M. Pooga, et al., FASEB Journal 12:67-77 (1998); D. Derossi, et al., Journal of Biological Chemistry 271:18188-18193(1996); G. Pietersz, et al., Vaccine 19:1397-1405 (2001); G. Elliott, and P. O'Hare, Cell 88:223-233 (1997); W. Derer, et al., FASEB Journal 16:132-133 (2002); E. Will, et al., Nucleic Acids Research 30:e59 (2002); J. Rothbard, et al., Journal of Medicinal Chemistry 45:3612-3618 (2002); L. Chen, et al., Chemistry & Biology 8:1123-1129 (2001); P. Wender, et al., Proceedings of the National Academy of Sciences of the United States of America 97:13003-13008 (2000)). The transport involves protein transduction domains (PTD) that are highly charged, short peptides (˜10 to 20 amino acids), containing basic amino acids (arginines and lysines), and that have the ability to form hydrogen bonds. The ability of PTD's to cross cell membranes is also concentration-dependent.
Multiple investigators have found that attachment of nucleic acids, peptides, and even large proteins to these PTD's will allow their transduction across all cell membranes in a highly efficient manner (S. Schwarze and S. Dowdy, Trends in Pharmacological Sciences 21 :45-48 (2000)). Three PTD's have been described which share the common characteristics of being potential DNA binding proteins: HIV-TAT, VP22, and Antennapedia (S. Schwarze, et al., Science 285:1569-1572 (1999); D. Derossi, et al., Journal of Biological Chemistry 271:18188-18193(1996); G. Elliott, and P. O'Hare, Cell 88:223-233 (1997)). Based on computer modeling and the prediction that these PTD's often have a strong a-helical character with a face of basically charged residues (arginines), investigators have begun to create synthetic peptides with greater ability to efficiently and quickly transduce across cell membranes. The exact mechanism of protein transduction is not known but is not receptor mediated and is independent of temperature making it unlikely that endocytosis or transporter mechanisms are involved (D. Mann and A. Frankel, EMBO Journal 10:1733-1739 (1991)). Furthermore, treatment of cells with drugs that inhibit cellular transport, such as brefeldin A (inhibits golgi transport), do not affect transduction of PTD's (G. Elliott, and P. O'Hare, Cell 88:223-233 (1997)).
Recently it was shown that the PTD derived from the HIV genome, HIV-TAT (trans-activator of transcription, “TAT”), has the ability to move attached peptides, large proteins, and nucleic acids across virtually all cell membranes, including brain, in a non-receptor mediated fashion (S. Schwarze, et al., Science 285:1569-1572 (1999); G. Cao, et al., Journal of Neuroscience 22:5423-5431 (2002); A. Gustafsson, et al., Circulation 106:735-739 (2002); H. Nagahara, et al., Nature Medicine 4:1449-1452 (1998)). The attached proteins are refolded into an active conformation once inside the cell and are biologically active. The full length TAT protein, originally described in 1988, by Green and Lowenstein, and is an 86 amino acid protein encoded by the HIV virus (S. Fawell, et al., Proc.Natl.AcadSci.U.S.A. 91:664-668 (1994); A. Frankel, and C. Pabo, Cell 55:1189-1193(1988); M. Green and P. Loewenstein, Cell 55:1179-1188(1988)). More specifically, an 11 amino acid arginine-and lysine-rich portion of the TAT sequence, YGRKKRRQRRR (SEQ ID NO:3), conjugated to peptides that do not normally cross membranes, is able to transduce across cell membranes and deliver a biologically active fusion protein to tissues. Furthermore, when a TAT-fusion protein was injected into mice for two weeks, there were no gross signs of neurological problems or system distress. Previously, TAT-fusion proteins were shown to be capable of delivering an active fusion protein that affects mitochondrial function, though in both cases, the fusion protein was not processed by the mitochondria. (G. Cao, et al., Journal of Neuroscience 22:5423-5431 (2002); A. Gustafsson, et al., Circulation 106:735-739 (2002)).
In summary, PTD's appear to offer a method for the efficient and rapid transport of highly charged, polar compounds across virtually all cell membranes and tissues in a concentration-dependent manner. This includes the mitochondrial membranes. These PTD's are well tolerated with only minimal detrimental effects seen at high concentrations in cell culture. However, because PTD-fusion proteins follow a concentration gradient, their use as therapeutic vehicles is limited by loss of the PTD-fusion protein from the cell unless the protein is bound or processed.